Alice:LabNotes/2008-12-15: Difference between revisions

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>Zsakura2
>Zsakura2
 
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*Ethanol purification and PAGE gel quantification.
*Ethanol purification and PAGE gel quantification.


  yield: 4ng/ul *80ul
  yield: 5ng/ul *80ul
  I ran a gel to quantify the gel along with the PCR product. Then I found that batch template 2 is not working.  
  I ran a gel to quantify the gel along with the PCR product. Then I found that batch template 2 is not working.  
  Therefore half of the plate is actually not working, thus final percent yield is too low.
  Therefore half of the plate is actually not working, thus final percent yield is too low.

Latest revision as of 21:03, 21 December 2008

CES27k9bp probe prep[edit]

Templates preparation[edit]

CES27K9bp-1 reaction system:                    x4
 E27kV2 (20nM):                         1ul         
 EconoTaq 2x master mix:               50ul       
 50X SYBG I                           0.4ul    
 100uM Ap1V4IU                        0.2ul     
 100uM AP2V4                          0.2ul
 H2O                                   50ul              

94C 2min -> (94C 45sec -> 60C 1min -> 72C 1min) x 18 -> 72C 3min -> 15C hold. Purified with Qiaquick columns. 
Result is 104nM dissovled in 50ul of H2O. Dilute it to 20nM with nonstick tube and 1X TE buffer. 
CES27K9bp-2 reaction system:                  x4
 CES27K 9bp (20nM):                     2ul         
 1.1X Platinum Taq SuperMix:           90ul       
 50X SYBG I                           0.6ul    
 100uM AP1V4IU                        0.4ul     
 100uM AP2V4                          0.4ul
 H2O                                    8ul              

94C 2min -> (94C 45sec -> 58C 1min -> 72C 1min) x 13 -> 72C 3min -> 15C hold. Purified with Qiaquick columns.
Result is 104nM dissovled in 60ul of H2O. Dilute it to 20nM with nonstick tube and 1X TE buffer.

PCR[edit]

  PCR system 1:                                  x48        
  Template (CES27K9bp-1):              0.1ul    9.6         
  EconoTaq 2x master mix:               50ul   2400
  50X SYBG I                           0.4ul   19.2    
  100uM AP1V41U                        0.1ul    4.8 
  100uM AP2V4                          0.1ul    4.8        
  H2O                                   50ul   2400   
  PCR system 2 has the same protocol except the use of CES27K9bp-2 template    
  PCR system 3:                                  x48
  Template (CES27K9bp-1):                0.2ul     9.6          
  EconoTaw 2X master mix                  50ul    2400
  50X SYBG I                             0.4ul    19.2   
  100uM AP1V41U                          0.1ul     4.8    
  100uM AP2V4                            0.1ul     4.8        
  H2O                                     50ul    2400
  PCR system 4:                                  x48
  Template (CES27K9bp-2):                0.4ul    19.2          
  EconoTaw 2X master mix                  50ul    2400
  50X SYBG I                             0.4ul    19.2   
  100uM AP1V41U                          0.1ul     4.8    
  100uM AP2V4                            0.1ul     4.8        
  H2O                                     50ul    2400

94C 2min -> (94C 45sec -> 58C 1min -> 72C 1min) x 22 -> 72C 5min -> 15C hold.
PCR result:
reaction system 3 & 4:
File:12-17-08 27k9bp.jpg

reaction system 1 & 2: (note that system 2 never shoot up because of not enough template added)
File:12-16-08 27k9bp.jpg

purified with ethanol precipitation, elute in 125ul H2O.

Lambda exo digestion[edit]

reaction system 1:

  • To 125ul of DNA, add 14ul 10X lambda exo buffer, 2ul lambda exo.
  • 37C 1 hr -> 75C 15 min
  • Purified the digestion Qiaquick column, eluted with 50ul ddH2O
  • Yield: 21.9ng/ul x 50ul 260/280=1.91


reaction system 3&4:

  • To 125ul of DNA, add 14ul 10X lambda exo buffer, 3ul lambda exo.
  • 37C 1 hr -> 75C 15 min
  • Purified the digestion Qiaquick columns, eluted with 80ul ddH2O, combine the elution.
Yield: 81.6ng/ul * 80ul            
      72.8ng/ul * 80ul                   
      260/280=1.90 for both

Dpn II & USER enzyme digestion[edit]

reaction system 1:

  • To 50ul of ssDNA, add 1.5ul USER enzyme, 37C 1hr.
  • Add 6ul 10X DpnII buffer, 2ul 100uM DpnII-V4 guide oligo
  • 94C5min -> 60C 5min -> turn off and wait for 20min -> add 1ul Dpn II (50K/ml)-> 37C 1h.
  • Add 1ul USER enzyme -> 37C 1h -> 75C 20min
  • PAGE purification in 6% TB-Urea 2D gels.
  • DNA were eluted from the shearing gel assembly and Nanosep columns.
  • Ethanol purification and PAGE gel quantification.

reaction system 3&4:

  • To 80ul of ssDNA, add 4ul USER enzyme, 37C 1hr.
  • Add 10ul 10X DpnII buffer, 2ul 100uM DpnII-V4 guide oligo
  • 94C5min -> 60C 5min -> turn off and wait for 20min -> add 2ul Dpn II (50K/ml) -> 37C 1h.
  • Add 2ul USER enzyme -> 37C 1h -> 75C 20min
  • PAGE purification in 6% TB-Urea 2D gels.
  • DNA were eluted from the shearing gel assembly and Nanosep columns.
  • Ethanol purification and PAGE gel quantification.
yield: 5ng/ul *80ul
I ran a gel to quantify the gel along with the PCR product. Then I found that batch template 2 is not working. 
Therefore half of the plate is actually not working, thus final percent yield is too low.